Activation of a G protein-coupled receptor (GPCR) triggers downstream signalling pathways whose identity is determined not only by the genetic background of the cell, but also by the interacting ligand. Assays that measure endogenous GPCR signalling in vivo are needed to specify the intracellular signalling pathways leading to therapeutic vs. adverse outcomes in animal models. To this end, genetically encoded biosensors can be expressed in vivo with cell type specificity to report GPCR signalling in real time. Biosensor imaging is facilitated by novel microscopic and photometric techniques developed for imaging in behaving animals. The techniques discussed here herald a new wave of in vivo signalling studies that will help identify therapeutically relevant signalling, and design functionally selective drugs for neuropsychiatric diseases.